qBiomarker Copy Number PCR Assays

用于分析特定位点的拷贝数变化和改变

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Cat. No. / ID: 337812

Laboratory-verified qPCR assays for measuring changes in copy number

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qBiomarker Copy Number PCR Assays 旨在用于分子生物学应用。这些产品不能用于疾病诊断、预防和治疗。

Configure at GeneGlobe

Find or custom design the right target-specific assays and panels to research your biological targets of interest.

Features

可选超过一千万个分析，覆盖基因组的各个区域
经湿法验证
简单real-time PCR操作流程
免费的网络数据分析软件

Product Details

qBiomarker Copy Number PCR Assays可对单个基因或目的区域（GOI或ROI）的拷贝数变化进行特定、准确、可重复、易解读的分析。每次分析都经实验验证，可即用于微阵列研究、特定靶标筛选和相关研究。

Performance

每个qBiomarker Copy Number PCR Assay都经湿法验证，有几个因素影响real-time PCR结果的准确性：特异性、宽动态范围和统一的高扩增效率。分析质量的实验室验证确保qBiomarker Copy Number PCR Assays获得可靠结果。

qBiomarker Copy Number PCR Assays可准确鉴定细胞学方法发现含有染色体畸变的细胞系中的非整倍体。检测X染色体上的 AR和 MECP2，可准备定量含有1、2、3、4拷贝X染色体细胞系的基因拷贝数。

See figures

qBiomarker Copy Number Assays accurately identify aneuploidy.

Principle

所有qBiomarker Copy Number PCR Assays都设计在基因组的独特区域。每个芯片都含有一个多拷贝参照分析，即qBiomarker Multicopy Reference Copy Number PCR Assay (MRef)。参照分析检测人体基因组中出现超过40次的稳定序列，其拷贝数不会受自身基因组变化影响或影响很小。检测包含参照分析可运用ΔΔC_T法对特定靶标进行准确的拷贝数分析或相对拷贝数变化分析。一体化、即用型qBiomarker SYBR^® Green PCR Mastermixes经优化，可提供最大化的位点特异性扩增并产生较少引物二聚体。

Procedure

从新鲜、冷冻或福尔马林固定石蜡包埋（FFPE）样本中分离基因组DNA。将每个样本与合适的即用型qBiomarker SYBR Green Mastermix和qBiomarker Copy Number PCR Assay混合。在另一管中，将每个样本与预混液和qBiomarker Multicopy Reference Copy Number PCR Assay混合。在real-time PCR仪上运行，使用免费的数据分析工具将检测样本与参照基因组对比，通过ΔΔC_T法确定拷贝数变化。

Applications

qBiomarker Copy Number PCR Assays非常适合准确检测新鲜、冷冻或福尔马林固定石蜡包埋（FFPE）样本的单个位点的拷贝数改变或变化。

Supporting data and figures

qBiomarker Copy Number PCR Assays accurately identify aneuploidy.

Resources

Download Safety Data Sheets for QIAGEN product components.

For real-time PCR-based, copy number alteration and variation analysis

For profiling copy number variation and alterations

For real-time PCR-based, copy number alteration and variation analysis

FAQ

Various sample types can be used on the arrays, including fresh frozen cell line and tissue samples, cell line admixtures, PAXgene fixed tissue samples, and FFPE tissue samples.

FAQ ID — 3423

The assays will work with heterogeneous samples. However, because the tumor samples are “diluted” by the normal cells with diploid genome, the absolute observed copy number for each locus will be an average of the tumor cells and normal cells. However, the p-value should give an indication as to whether there is a statistically significant amplification or deletion that happens in the cell population for the locus of interest.

FAQ ID — 3419

qBiomarker Copy Number PCR Arrays and Assays are compatible with fixed samples. However, when fixed samples are analyzed, the user is strongly recommended to refer to the “Important Points Before Starting” section and “Appendix A: Quality Control of Genomic DNA Using the DNA QC Plate” in the qBiomarker Copy Number PCR Array Handbook for considerations on selecting an appropriate calibrator sample(s), if available.

FAQ ID — 3416

Having the assays in quadruplicate will enable accurate copy number call via statistical analysis (implemented in online data analysis software).

FAQ ID — 3414

The qBiomarker SYBR ROX Mastermix is suitable for use with the following real-time cyclers: Applied Biosystems models 5700, 7000, 7300, 7500 (Standard and Fast), 7700, 7900HT (Standard and Fast 96-well block, 384-well block), StepOnePlus, ViiA 7 (Standard and Fast 96-well block, 384-well block); Eppendorf Mastercycler ep realplex models 2, 2S, 4, 4S; Stratagene models Mx3000P, Mx3005P, Mx4000; Takara TP-800.

The qBiomarker SYBR Fluor Mastermix is suitable for use with the following real-time cyclers: Bio-Rad models iCycler, iQ5, MyiQ, MyiQ2.

The qBiomarker SYBR ROX FAST Mastermix is suitable for use with the Applied Biosystems models 7000, 7300, 7500 (Standard and Fast), 7700, 7900HT (Standard and Fast 96-well block, 384-well block), StepOnePlus; ViiA 7 (Standard and Fast 96-well block, 384-well block); Eppendorf Mastercycler ep realplex with or without ROX filter set; Stratagene models Mx3000, Mx3500, Mx4000; Takara TP-800; Rotor-Gene Q (QIAGEN), and Rotor-Gene 6000.

FAQ ID — 3422

Yes.

FAQ ID — 3425

Data analysis uses the ΔΔC_T method. At the qBiomarker Copy Number PCR Array and Assay Data Analysis Web portal (http://www.qiagen.com/products/genes and pathways/data analysis center overview page), C_T data can be entered and the Web-based software will automatically perform quantification.

FAQ ID — 3421

Sample heterogeneity can lead to lower-than-expected copy number calls. In the presence of non-tumor cells that have normal diploid genomes, the copy number call is dependent on the copy number of the target gene in the cancer cells and the amount of non-tumor cells in the heterogeneous sample. If the percentage of non-tumor cells in the sample can be estimated, the copy number for a gene can be estimated based on Table 16 in the user manual.

FAQ ID — 3413

One or more of the following 3 issues can lead to high C_T values for all wells:

1. The cycling program is incorrect. Please make sure to program the real-time PCR cycler with the temperature profile shown in the protocols.

2. DNA quality is poor. Check the DNA quality using the DNA QC Plate (see the qBiomarker Copy Number PCR Array Handbook) or on an agarose gel to see if the DNA is degraded. If the DNA is not degraded, it could be of insufficient purity. We recommend using one of the kits indicated in Table 3 of the qBiomarker Copy Number PCR Array Handbook for isolation of high-quality DNA.

3. Too little DNA is used. Make sure that the DNA has been properly quantified. During the DNA purification process, it is essential to perform an RNase digestion. RNA contamination in the DNA sample will lead to overestimation of DNA quantity. Note that larger amounts of DNA are recommended when working with FFPE samples.

FAQ ID — 3412

DNA concentration and purity can be measured by UV spectrophotometry.

Dilute samples and measure absorbance in 10 mM Tris•Cl, pH 8.0. An absorbance reading of 1.0 at 260 nm in a 1 cm detection path corresponds to a DNA concentration of 50 µg/ml. All DNA samples should meet the following criteria:

1. Concentration, as measured by A260, should be greater than 10 µg/ml
2. A260/A280 ratio should be greater than 1.8
3. A260/A230 ratio should be greater than 1.7

It is also strongly recommended that DNA quality be measured with the DNA QC Plate.
DNA quality and consistency can be checked more reliably with the DNA QC Plate by real-time PCR measuring 7 reference genes. For a detailed procedure, see the qBiomarker Copy Number PCR Array Handbook.

FAQ ID — 3424

Header
Format	Genomic DNA
96-well plate / Rotor-Disc (per reaction)	4 ng (fresh); 8–20 ng (FFPE)
384-well plate (per reaction)	2 ng (fresh); 4–10 ng (FFPE)

FAQ ID — 3418

DNA from fresh frozen samples can be subjected to whole genome amplification (WGA) before use in downstream copy number PCR analysis. The recommended method is QIAGEN’s REPLI-g or REPLI-g UltraFast Kits. For DNA from FFPE samples, we do not recommend amplification before copy number PCR analysis.

FAQ ID — 3415

In general, the genes on each qBiomarker Copy Number Array are selected from primary literature and public databases based on their amplification and deletion frequency in a disease or pathway, function in cancer or complex disease/trait signaling pathways, and their association with the disease phenotype or progression.

FAQ ID — 3420